Figure 5.

Detection of essential influenza virus receptors entry factors in organoids and infection by influenza pseudotype H5N1 and viral replication by H7N1 virus. (A) Sialic acids (SAs) of cell surface glycoproteins and glycolipids are the receptors for influenza virus. The presence of SAs and galactose on bronchiolar (BRON, left histograms) and alveolar (ALV, right histograms) were detected by flow cytometry using the fluorescein-conjugated lectins, MAL I, recognizing galactose, and SNA, recognizing α 2,6-linked sialic acids. CTRL histogram represents the unstained control cells. The representative flow cytometry histograms shown here were from stained dissociated organoids from patient L2. (B) In situ detection of cells infected by the pseudotyped luciferase expressing H5N1 influenza virus was done by immunostaining to detect luciferase (LUC) and keratin 5 (KRT5) in formalin fixed paraffin embedded (FFPE) sections of infected organoids. Organoids of BRON (upper panel) and ALV (lower panel) organoid cultures are shown. Sections are counterstained with DAPI. Scale bar in all images equals 100 µm. (C) Organoids were infected with the replicating influenza virus H7N1 strain at an estimated multiplicity of infection (MOI) of 7. Plaque assay was used to confirm efficient replication of pseudotyped influenza virus in the organoid cultures. Infected MDCK cells were used as a positive control in this assay. Plaques are visible in MDCK cell cultures inoculated with conditioned media harvested from infected bronchiolar and alveolar organoid cultures, as well as conditioned media harvested from the positive control MDCK cells. Conditioned media from uninfected controls showed no sign of plaque formation (lower row). Counterstaining by crystal violet. Magnification equals 4x.